Summary

Whereas most phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity is
localized in the cytoplasm, PtdIns 4-kinase activity has also been detected in
membranedepleted nuclei of rat liver and mouse NIH 3T3 cells. Here we have
characterized the PtdIns 4-kinase that is present in nuclei from NIH 3T3
cells. Both type II and type III PtdIns 4-kinase activity were observed in the
detergent-insoluble fraction of NIH 3T3 cells. Dissection of this fraction
into cytoplasmic actin filaments and nuclear lamina-pore complexes revealed
that the actin filament fraction contains solely type II PtdIns 4-kinase,
whereas lamina-pore complexes contain type III PtdIns 4-kinase activity. Using
specific antibodies, the nuclear PtdIns 4-kinase was identified as PtdIns
4-kinase β. Inhibition of nuclear export by leptomycin B resulted in an
accumulation of PtdIns 4-kinase β in the nucleus. These data demonstrate
that PtdIns 4-kinase β is present in the nuclei of NIH 3T3 fibroblasts,
suggesting a specific function for this kinase in nuclear processes.

Introduction

The metabolism of phosphoinositides is known to play a crucial role in the
transduction of signals triggered by a variety of hormones and growth factors.
In particular, phosphatidylinositol(4,5)bisphosphate
(PtdIns(4,5)P2) has been shown to be involved in the
formation of stress fibers and focal adhesions via interactions with several
actin-binding proteins, including profilin, gelsolin and N-WASP
(
Isenberg and Niggli, 1998).
In addition, products of phosphoinositide 3-kinase (PI 3-kinase),
PtdIns(3)P, PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 have been shown to act as lipid second
messengers by binding to specific protein domains such as the Pleckstrin
Homology, Phox- and FYVE domains and have been implicated in many
intracellular processes such as intracellular trafficking, mitogenesis and
actin rearrangements (
Fruman et al.,
1999;
Kanai et al.,
2001;
Lemmon and Ferguson,
1998). An important precursor in both signaling pathways is
PtdIns(4)P, which is synthesized by phosphatidylinositol 4-kinase
(PtdIns 4-kinase).

PtdIns kinases from mammalian cells have been divided into three types
(
Endemann et al., 1987;
Whitman et al., 1987). Type I
is a PI 3-kinase, which is strongly inhibited by non-ionic detergents such as
Triton X-100 (
Whitman et al.,
1988). Type II and type III PtdIns kinases are both PtdIns
4-kinases and are activated by non-ionic detergents. Different inhibitors can
be used to discriminate between type II and III PtdIns 4-kinases. Adenosine
inhibits only type II PtdIns 4-kinase, whereas both the PI 3-kinase inhibitors
wortmannin and LY294002 inhibit only type III PtdIns 4-kinase. The latter
requires a much higher concentration than for inhibition of PI 3-kinase
(
Downing et al., 1996).

We have previously reported that PtdIns 4-kinase activity is present in the
detergent-insoluble fraction and membranedepleted nuclei of NIH 3T3 cells
(
Payrastre et al., 1992;
Payrastre et al., 1991).
Further fractionation of the nuclei showed that PtdIns 4-kinase activity was
specifically associated with the lamina-pore complex, whereas
PtdIns(4)P 5-kinase activity was detected in the internal matrix
fraction (
Payrastre et al.,
1991). The importance of a nuclear inositol lipid metabolism is
stressed by the finding that nuclear phosphoinositide levels were found to
decrease during the S-phase of the cell cycle
(
York and Majerus, 1994).
Furthermore, in Friend erythroleukaemia cells it was shown that insulin growth
factor-1 (IGF-1) stimulates the hydrolysis of nuclear but not cytoplasmic
phosphoinositides (
Divecha et al.,
1991). Recently, a large increase in nuclear PtdIns(5)P
mass was observed during the G1 phase of murine erythro leukaemia cells, which
suggests a role for this lipid in cell proliferation
(
Clarke et al., 2001).

In this paper we have characterized the PtdIns 4-kinase activity that is
present in the detergent-insoluble fraction and membrane-depleted nuclei of
NIH 3T3 fibroblasts. We show that a mixture of type II and type III PtdIns
4-kinase activity is bound to the detergent-insoluble fraction of NIH 3T3
cells. Further dissection of this fraction revealed that the PtdIns 4-kinase
activity in the actin filament fraction is predominately type II whereas the
PtdIns 4-kinase activity present in isolated lamina-pore complexes is type
III. Fractionation studies indicate that the nuclear type III kinase is PtdIns
4-kinase β (PI4Kβ). The localization of PtdIns 4-kinase β in
the nucleus suggests a specific role for this PtdIns 4-kinase in the nuclear
inositol cycle.

Materials and Methods

Materials

The antibody directed against lamin A/C (41CC4) was a generous gift of R.
van Driel (E.C. Slater Institute, University of Amsterdam, The Netherlands),
and the antibody against PI4Kβ was a generous gift of Rachel Meyers (MIT,
Boston, USA) (
Meyers and Cantley,
1997). The monoclonal antibody against actin was from Amersham
(Amersham International, UK), the antibodies against caveolin-1 and against GM
130 were from R&D Systems (Minneapolis, USA), the antibody against
myc-epitope 9E10 was from Roche Molecular Biochemicals (Germany) and the
monoclonal antibody against tubulin was from Calbiochem (San Diego, USA). The
secondary antibodies donkey anti-mouse-horse radish peroxidase and donkey
anti-rabbit-horse radish peroxidase were from Jackson ImmunoResearch
Laboratories Inc. (West Grove, USA). Leptomycin B and lipids were obtained
from Sigma Co. (St. Louis, USA), and [γ-32P]-ATP was from
Amersham. Restriction enzymes were obtained from Roche Molecular
Biochemicals.

Construction of eukaryotic expression vector for epitopetagged
PI4K230 and PI4Kβ

The human PI4K230 cDNA was a kind gift from Thor Gehrmann, and details of
it have been published previously
(
Gehrmann et al., 1999). The
cDNA was cut out of the pFastBac HTa vector (GibcoBRL, Paisley, UK) using
EcoRI and XhoI and cloned into the pcDNA3.1 zeo+ vector
(Invitrogen, USA). The cDNA was tagged with the myc-epitope (MEQKLISEEDL)
using an oligo-linker with restriction sites HindIII and
EcoRI. The human PI4Kβ cDNA was obtained by PCR from the human
brain QUICK-Clone TM cDNA library (Clontech, USA) using the Expand
High-Fidelity System (Roche Molecular Biochemicals, Germany). Primers were
designed on the basis of the sequence of PI4Kβ
(
Meyers and Cantley, 1997),
with the restriction sites XhoI (forward primer) and XbaI
(reverse primer). The cDNA was cloned into the pcDNA3.1 zeo+ vector
(Invitrogen, USA) and tagged with the coding sequence of the myc epitope
(MEQKLISEEDL) using an oligo-linker with restriction sites KpnI and
XhoI.

Production of monoclonal antibody against PI4K230

The DNA fragment coding for amino acids 1691-1779 of PI4K230 was obtained
by PCR from the human brain QUICK-CloneTM cDNA library (Clontech, USA) using
the Expand High-Fidelity System (Roche Molecular Biochemicals, Germany). This
fragment was cloned into the pGEX-2T vector (Amersham, UK). After
transformation of this vector into DH5α, a GST fusion protein was
isolated and cut with thrombin as described previously
(
Whitehead et al., 1999). Mice
were injected with 50 μg of purified protein mixed (1:1) with Freund's
complete adjuvants; the GST fusion protein was used for further screening.
Production and isolation of the monoclonal antibody against PI4K230 was
performed according to standard procedures
(
Schulz et al., 1987).

Isolation of lamina-pore complexes

Membrane-depleted nuclei were isolated from NIH 3T3 fibroblasts according
to a slightly modified procedure of Payrastre et al.
(
Payrastre et al., 1992). To
isolate lamina-pore complexes, the membrane-depleted nuclei were incubated
with DNaseI (200 units/107 nuclei) for 30 minutes at 37°C in
hypotonic buffer (20 mM Hepes, pH 7.4, 10 mM KCl, 3 mM MgCl2, 1 mM
Na3VO4, 1 mM benzamidine, 1 mM PMSF).
(NH4)2SO4 was added to a final concentration
of 0.25 M. After 15 minutes on ice the nuclear matrices were centrifuged at
1000 g for 5 minutes and washed once with hypotonic buffer
without detergent. In order to remove the internal matrix, the nuclear
matrices were incubated with 0.25 M (NH4)2SO4
and 40 mM dithiothreitol (DTT) for 20 minutes at 37°C. The peripheral
matrices were centrifuged at 10,000 g for 5 minutes, washed
with ice cold hypotonic buffer without detergent, resuspended in the same
buffer and used immediately.

Lipid kinase assay

Lipid kinase activities were measured as previously described by Payrastre
et al. (
Payrastre et al.,
1991) in a final volume of 200 μl containing 50 mM Tris-HCl (pH
7.4), 10 mM MgCl2, 50 μM ATP, 10 μCi [γ-32P]
ATP, 0.3% Triton X-100, 0.2 mg/ml PtdIns/PtdChol vesicles (2:1) and 30-50μ
g protein. Proteins were sonicated three times for 10 seconds on ice prior
to use. The inhibitors adenosine (200 μM), wortmannin (1 μM) or LY294002
(10 μM) were added to the reaction mixture when indicated. The reaction was
started by adding the PtdIns/PtdChol vesicles and carried out at 37°C for
20 minutes under constant shaking. Under these conditions, lipid synthesis was
linear with protein concentration and with time. The reaction was stopped by
adding 400 μl chloroform/methanol (1:1) and the lipids were immediately
extracted following the modified method of Bligh and Dyer
(
Bligh and Dyer, 1959;
Payrastre et al., 1991).
Phosphoinositides were separated by thin layer chromatography (TLC) on
silica-gel-coated glass plates using chloroform/methanol/4.3 M
NH4OH (90/70/20) as a solvent
(
Gonzales-Sastre and Floch-Pi,
1968). TLC plates were analyzed using a phosphoimager
(Phosphoimager SI™, Molecular Dynamics Inc.), and the spots were
quantified using ImageQuaNT software.

Gel electrophoresis and western blotting

Protein concentrations were determined according to Peterson
(
Peterson, 1977). Protein
samples were resuspended in sample buffer (60 mM Tris-HCl, 10% glycerol (v/v),
45 mM dithiothreitol, 80 mM sodium dodecyl sulfate, pH 6.8), separated by
SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked using 3%
milk powder (Protifar, Nutricia, Zoetermeer, The Netherlands) in TBST (20 mM
Tris-HCl pH 7.4, 150 mM NaCl and 0.1% Tween-20) for 45 minutes and incubated
with the primary antibody diluted 1:5,000 (or 1:40,000 for PI4Kβ) in 0.3%
milk powder in TBST for 1 hour. The membrane was washed four times in the same
buffer for 5 minutes each and subsequently incubated with the secondary
antibody (donkey anti-mouse-horse radish peroxidase, or donkey
anti-rabbit-horse radish peroxidase for PI4Kβ) diluted 1:10,000 in 0.3%
milk powder in TBST for 45 minutes. The blot was washed three times with the
same buffer for 5 minutes and once for 5 minutes in TBST. Proteins were
detected using the chemiluminescence procedure as described by the
manufacturer (Lumilight Plus, Roche Diagnostics), and visualized using a
lumino-imager (FluorS, BioRad)

Immunofluorescence

COS-1 cells were seeded on 15 mm cover slips and, when indicated,
transiently transfected with myc-PI4Kβ using FuGENE 6 transfection
reagent (Roche Molecular Biochemicals, Germany) according to the manufacturer.
Cells were fixed for 20 minutes in 4% formaldehyde, washed twice with PBS,
followed by a 5 minutes incubation with 0.2% Triton X-100 in PBS. Cover slips
were washed twice with PBS followed by a 10 minutes quenching of formaldehyde
using 50 mM Glycine in PBS. After washing twice with 0.2% Gelatin in PBS (PBG)
cells were incubated for 60 minutes with anti-myc antibody (9E10, Roche
Molecular Biochemicals), at a concentration of 800 ng/ml in PBG. After washing
four times for 5 minutes, cells were incubated with GAM-Cy3 (Jackson
ImmunoResearch) at a concentration of 1.3 μg/ml in PBG for 45 minutes.
After washing four times with PBG and once with PBS, cells were imbedded in
mowiol/PPD and analyzed by confocal microscopy.

Results

PtdIns 4-kinase activity in the detergent insoluble fraction of NIH
3T3 fibroblasts is a mixture of type II and type III activity

To discriminate between type II and III PtdIns 4-kinases we used the PtdIns
kinase inhibitors adenosine and wortmannin. Adenosine specifically inhibits
type II PtdIns 4-kinase activity, whereas wortmannin can be used to inhibit
type III PtdIns 4-kinase activity. In total cell lysates, PtdIns 4-kinase
activity was significantly inhibited by 200 μM adenosine
(
Fig. 1A). In contrast, no
effect of wortmannin was observed, which indicates that the majority of the
PtdIns 4-kinase activity in total cell lysates is a type II PtdIns 4-kinase.
To exclude any contribution of PI 3-kinase activity, the non-ionic detergent
Triton X-100 was added to the lipid kinase assay, which inhibits PI 3-kinase
activity. This was controlled by adding a low concentration of LY294002, and
this inhibitor did not reduce PtdIns kinase activity any further.

Lipid kinase activity in total cell lysate and detergent insoluble
fraction. (A) Total cell lysate (10 μg) and detergent insoluble fraction
(DIF) (50 μg) from NIH 3T3 cells were applied to an in vitro lipid kinase
assay using PtdIns as substrate. After an incubation of 20 minutes at
37°C, the lipids were extracted, separated on TLC and the
PtdIns(4)P spots were quantified (total cell lysates, s.e.m.,
n=3; DIF, s.e.m., n=4) (*P<0.01).
Adenosine, 200 μM; wortmannin, 1 μM; LY294002, 10 μM. (B) Total cell
lysates (10 μg) from NIH 3T3 cells were incubated with increasing amounts
of adenosine and applied to an in vitro kinase assay. Results are expressed as
the percentage of control activity without adenosine.

We next analyzed the PtdIns 4-kinase activity that is present in the
detergent-insoluble fraction (DIF) of NIH 3T3 cells. This fraction was
isolated by a treatment of the cells with the non-ionic detergent Triton
X-100. The fraction of total PtdIns 4-kinase activity that is associated with
the DIF of NIH 3T3 cells was approximately 11% of total PtdIns 4-kinase
activity. Analysis of the PtdIns kinase activity that is associated with the
detergent insoluble fraction showed no inhibition by adenosine, but this time
a high concentration of wortmannin inhibited the lipid kinase activity by more
than 60% (
Fig. 1A). Again, no
significant inhibition by a low concentration of LY294002 was observed. These
observations demonstrate that the DIF of NIH 3T3 cells contains both type II
and type III PtdIns 4-kinase activity.

An unexpected finding was that the type II PtdIns 4-kinase activity in
total cell lysates could not be completely inhibited by adenosine. This
observation suggests the existence of an adenosine-insensitive PtdIns 4-kinase
activity. In order to investigate this possibility we analyzed the effect of
higher concentrations of this inhibitor on PtdIns 4-kinase activity in total
cell lysates. Adenosine at a concentration of 1 mM inhibited PtdIns 4-kinase
activity by 90%, which indicates that the existence of an
adenosine-insensitive pool is highly unlikely
(
Fig. 1B).

The DIF is composed of cytoskeletal structures and membrane-depleted
nuclei. In order to determine the location of the two types of PtdIns
4-kinases within the DIF, we dissected this fraction into cytoplasmic actin
filaments and nuclear lamina-pore complexes. Actin filaments were isolated
using an in vitro depolymerization/repolymerization reaction as described
previously (
Payrastre et al.,
1991). Lamina-pore complexes were isolated from membrane-depleted
nuclei according to Payrastre et al.
(
Payrastre et al., 1992). The
purity of the nuclei was investigated enzymatically using
5′-nucleotidase, lactate dehydrogenase and antimycin A-insensitive
NADH-cytochrome c reductase as markers for the plasma membrane, cytoplasm and
endoplasmatic reticulum respectively, and their activity was found to be less
than 1% in the nuclear fraction (
Payrastre
et al., 1992).

Both fractions were analyzed by western blotting using antibodies against
different proteins that are characteristic for these fractions.
Anti-caveolin-1 was used as a marker for the DIF, anti-GM130 as a marker for
the Golgi apparatus, anti-actin as a marker for the cytoskeleton, and
anti-lamin was used as a marker for the nuclear matrix. The DIF marker
caveolin-1 is clearly present in the total cell lysate and in the DIF but
strongly reduced in nuclear and actin filament fractions
(
Fig. 2). The Golgi marker
GM130 is present in the total lysate but greatly reduced in the DIF.
Furthermore, this marker is absent in the fractions containing actin filaments
and nuclear matrix, indicating that these two fractions were not contaminated
by Golgi proteins. The nuclear marker lamin is clearly present in the DIF and
nuclear matrices and completely absent in the actin filament fraction. Lamin
was also detected in the total cell lysate, but the intensity of signal for
lamin was, for unknown reasons, less than that observed in the DIF. The
cytoskeletal marker actin is present in the total cell lysate, the DIF and in
the actin filaments. A small amount of actin is, however, also detectable in
nuclear matrix fraction, suggesting the presence of nuclear actin or a small
contamination of the nuclei by this cytoskeletal protein. By changing the
isolation conditions we have tried to optimise the nuclear isolation
procedure, but changes in the used detergent (NP40) or differences in salt
conditions did not result in the extraction of actin (data not shown). This
observation suggests that actin is a component of the nuclear matrix, an
observation that is in agreement with previous studies (for a review, see
Rando et al., 2000).

Subsequently, we tested the effects of the different lipid kinase
inhibitors on the PtdIns 4-kinase activity in these fractions. In contrast to
wortmannin, adenosine significantly inhibited PtdIns 4-kinase activity present
in the actin filament fraction (
Fig.
3). In the nuclear fraction, however, a high concentration of
wortmannin has a strong inhibitory effect on the formation of radioactive
PtdIns(4)P, whereas adenosine had no significant effect on the PtdIns 4-kinase
activity. These data demonstrate that isolated actin filaments contain type II
PtdIns 4-kinase activity, whereas the nuclei contain PtdIns 4-kinase activity
belonging to the type III family of PtdIns 4-kinases.

PI4Kβ is the nuclear type III PtdIns 4-kinase

At present, two type III PtdIns 4-kinases have been described: PI4K230 and
PI4Kβ (
Gehrmann et al.,
1999;
Meyers and Cantley,
1997). In order to determine which of these PtdIns 4-kinases is
present in NIH 3T3 cells we used specific antibodies against these two type
III PtdIns 4-kinases. To perform these experiments we raised a monoclonal
antibody against amino acids 1691-1779 of PI4K230 as described in the
Materials and Methods. The polyclonal antibody against PI4Kβ was obtained
from R. Meyers (MIT, Boston, USA) (
Meyers
and Cantley, 1997). The monoclonal antibody reacted strongly with
a myctagged PI4K230, which was expressed in COS-1 cells
(
Fig. 4). Remarkably, the
anti-PI4K230 recognized several bands, whereas anti-myc showed a positive
reaction with a single band. This is probably caused by N-terminal
degradation, as an N-terminal myc-epitope was used. Furthermore, no positive
reaction of the monoclonal anti-PI4K230 was found in lysates from
mock-transfected COS-1 or NIH 3T3 cells. In addition, no signal was observed
when this antibody was used with immunofluorescence microscopy on NIH 3T3, BHK
or CHO cells (data not shown). In contrast, PI4Kβ could easily be
detected in lysates from both COS-1 and NIH 3T3 cells
(
Fig. 4). From these results we
conclude that from the currently identified type III kinases only PI4Kβ
and not PI4K230 is present in COS-1 and NIH 3T3 cells.

PI4K230 is absent in NIH 3T3 and COS-1 cells. Cells were either mock
transfected or transfected with cDNA encoding myctagged PI4K230. Proteins from
cell lysates from equal amounts of cells were separated on 6% SDS-PAGE for
PI4K230 and on 8% SDS-PAGE for PI4Kβ and subsequently blotted and
incubated with antibodies directed against myc epitope (9E10), PI4K230 and
PI4Kβ. Anti-PI4K230 was prepared as described in the Materials and
Methods. Epitope-tagged PI4K230 was used as a control to demonstrate the
expression of PI4K230 in COS-1 cells.

Subsequently, we analyzed whether PI4Kβ is present in the nuclei of
NIH 3T3 and CHO cells. Membrane-depleted nuclei were isolated, and proteins
from total cell lysates, supernatant and nuclei were analyzed by western
blotting. Anti-tubulin was used as a marker for the cytoplasm, and only a
minor band was observed in the nuclear fraction
(
Fig. 5). PI4Kβ is clearly
present in the nuclear fraction of both NIH 3T3 and CHO cells. In addition,
CHO cells transiently transfected with epitope (myc)-tagged PI4Kβ,
leading to a higher expression level of PI4Kβ, shows a clear band
representing PI4Kβ in the nuclear fraction
(
Fig. 5).

The presence of PI4Kβ in the nuclear fraction. Proteins from cell
equivalents of cell lysate (CL), post nuclear supernatant (PNS) and five times
cell equivalents of membrane-depleted nuclei (N) were separated on 8% SDS-PAGE
and subsequently western blotted and incubated with antibodies directed
against tubulin, PI4Kβ and the myc-epitope (9E10). 3T3, proteins from
mock-transfected NIH 3T3 cells; CHO, proteins from mock-transfected CHO cells,
and CHO + mPI4Kβ, CHO cells transfected with epitope-tagged PI4Kβ
(myc).

PI4Kβ accumulates in the nucleus upon inhibition of nuclear
export

To obtain further proof of the nuclear localization of PI4Kβ, we
performed immunofluorescence microscopy on normal COS-1 cells, and COS-1 cells
transfected with epitope-tagged PI4Kβ. A similar distribution of
endogenous and transiently expressed PI4Kβ was found. PI4Kβ is
mainly located in the cytoplasm and is concentrated at the Golgi complex. Only
a weak staining of the nuclei was observed
(
Fig. 6A). A similar
distribution of epitope-tagged PI4Kβ was observed in transiently
transfected COS-1 cells (
Fig.
6C). Sequence analysis of PI4Kβ showed that this kinase also
contains two nuclear localization signals and a leucine-rich sequence
resembling a nuclear export signal (NES). This type of NES is involved in the
nuclear export of proteins regulated by Crm1, which can be inhibited by
leptomycin B (
Fornerod et al.,
1997;
Ossareh-Nazari et al.,
1997). Control and transfected COS-1 cells were treated for 16
hours with 10 ng/ml leptomycin B, fixed and immunostained for PI4Kβ. As
shown in
Figure 6B, leptomycin
B induced an accumulation of endogenous PI4Kβ in the nucleus. No staining
is observed in nucleoli. Similar results were obtained with COS-1 cells that
were transiently transfected with epitope-tagged PI4Kβ
(
Fig. 6D). These data
demonstrate that PI4Kβ is exported from the nucleus in a Crm1-dependent
way. Moreover, this indicates that in vivo PI4Kβ translocates to the
nucleus, supporting our biochemical data that PI4Kβ is present in the
nucleus.

PI4Kβ accumulates in the nucleus upon leptomycin B treatment. COS-1
cells were mock transfected (A,B) or transfected with myc-PI4Kβ (C,D).
Cells were left untreated (control, A,C) or treated for 16 hours with 10 ng/ml
leptomycin B (+LB, B,D). After incubation, cells were fixed and stained with
anti-PI4Kβ followed by GAR-Cy3 (A,B) or with an anti-myc antibody (9E10)
followed by GAM-Cy3 (C,D).

Discussion

In this paper we have characterized the PtdIns 4-kinases that are present
in the DIF and membrane-depleted nuclei of mouse NIH 3T3 cells. Although total
cellular PtdIns 4-kinase activity could not be inhibited by wortmannin, the
DIF contained a PtdIns 4-kinase activity that was significantly inhibited by
wortmannin. This indicates that the majority of total PtdIns 4-kinase activity
belongs to the type II kinases and, furthermore, that the DIF contains type
III PtdIns 4-kinase activity. Further dissection of the DIF into a
cytoskeletal and nuclear fraction revealed that the actin filament fraction
predominantly contains type II PtdIns 4-kinase activity, whereas the nuclear
PtdIns 4-kinase activity almost completely consists of type III PtdIns
4-kinase activity.

An important question is which of the known PtdIns 4-kinase isoforms is
responsible for the observed kinase activities in the different fractions.
Type II PtdIns 4-kinase is indicated as the PI4K55 and has recently been
cloned by two independent groups (
Barylko
et al., 2001;
Minogue et al.,
2001). This type II PtdIns 4-kinase binds tightly to the membrane
and bears little similarity to other known lipid or protein kinases. Two
mammalian type III PtdIns 4-kinase isoforms have been described: PI4Kβ,
the orthologue of PtdIns 4-kinase from the budding yeast Saccharomyces
cerevisiae Pik1p, and PI4K230, the mammalian orthologue for the yeast
isoform Stt4p (
Gehrmann and Heilmeyer Jr.,
1998;
Meyers and Cantley,
1997). Interestingly, Pik1p was found to bind to the nuclear pores
of yeast nuclei, whereas Stt4p was strictly located in the cytoplasm
(
Flanagan et al., 1993;
Yoshida et al., 1994). Both
the human and bovine PI4K230 have been sequenced, and this isoform contains
besides an SH3 and PH domain, two nuclear localization signals (NLS). Also
PI4Kβ contains two NLSs and one NES, which are all present in the
N-terminal part of the protein.

To identify the type III isoform present in the nucleus we first analyzed
which of these PtdIns 4-kinases is present in NIH 3T3 and COS-1 cells. Using
specific antibodies against both isoforms we could not detect PI4K230 in these
cells. This observation is in accordance with the work of Gehrmann and
colleagues who have shown that PI4K230 is mainly expressed in the brain
(
Gehrmann et al., 1999). In
contrast, western blot analysis of NIH 3T3 and COS-1 cells clearly showed the
presence of this kinase in these cells. Interestingly, endogenous PI4Kβ
was also present in membrane-depleted nuclei from NIH 3T3 cells. An increase
in nuclear presence was found when epitope-tagged PI4Kβ was expressed in
CHO cells. The nuclear presence of PI4Kβ is not caused by contamination
of the nuclear fraction by cytosolic proteins, as the nuclear preparations
were negative for tubulin and the cis-Golgi marker GM130.

Previously, PI4Kβ has been localized by fluorescence microscopy, and
it was predominantly found at the Golgi complex
(
Wong et al., 1997). In our
experiments we also observed a very weak staining of the nucleus, which is in
agreement with the low level of nuclear PI4Kβ, as determined with the
western blotting experiments. The nuclear PtdIns 4-kinase is, however, shown
to bind to the lamina-pore complex, which may be difficult to discern in the
fluorescent light microscope (
Payrastre et
al., 1992). PI4Kβ also contains, next to the two NLSs, a
leucine-rich sequence resembling a nuclear export signal (NES). Nuclear export
of a leucine-rich NES is regulated by Crm1, which can be blocked by leptomycin
B (
Ossareh-Nazari et al.,
1997). Inhibition of nuclear export by leptomycin B resulted in an
accumulation of PI4Kβ in the nucleus. PI4Kβ remained excluded from
nucleoli. This suggests that PI4Kβ is shuttling between the cytoplasm and
the nucleus in a Crm1-dependent process. Our previous results showed that
PtdIns 4-kinase activity is associated with the lamina-pore complex
(
Payrastre et al., 1992). It
is tempting to suggest that a small fraction of PI4Kβ that is transported
into the nucleus remains in the nucleus as a result of its binding to a
component of the nuclear-pore complex. This intranuclear location of
PI4Kβ is to be expected, because at this location it is in proximity to
its substrate PtdIns, which was recently shown to be present in the nuclear
envelope (
Vann et al., 1997).
The component of the lamina-pore complex that is responsible for the binding
of PI4Kβ remains to be elucidated.

The nuclear fraction of PI4β activity may fulfil a role in nuclear
inositol signaling (for a review, see
Irvine, 2000). Stimulation of
the cell with growth factors such as EGF and PDGF did not stimulate this lipid
kinase (P.d.G, unpublished). On the other hand, the translocation of
PI4Kβ into the nucleus may result in the stimulation of the nuclear
phosphoinositide cycle by inducing the generation of PtdIns(4)P,
which is the substrate of the nuclear type I PtdIns(4)P 5-kinase. We
have previously shown that this PtdIns P kinase activity is located in the
inner nuclear matrix, which is consistent with the localization of the type I
PtdIns(4)P 5-kinase in nuclear speckles
(
Boronenkov et al., 1998).
Components of the mRNA machinery are also located in these nuclear speckles,
suggesting a function for the nuclear PtdIns metabolism in mRNA processing. On
the other hand, the products of PtdIns 4-kinase, PtdIns(4)P and
inositol(1,4)bisphosphate may have a function themselves, as they are known to
activate a low specificity form of DNA polymerase α in vitro
(
Sylvia et al., 1988).
Moreover, the level of PtdIns(5)P was recently shown to increase
dramatically during the G1 phase of murine erythro leukemia cells
(
Clarke et al., 2001). These
data all point to a function for the nuclear inositol cycle in the regulation
of cell proliferation and cell differentiation.

In conclusion, the results shown in this paper reveal that type II PtdIns
4-kinase activity is associated to actin filaments, whereas type III PtdIns
4-kinase activity is present in nuclei. The type III PtdIns 4-kinase was
identified as the PI4Kβ. Studies using leptomycin B suggest that the
nuclear levels of PI4Kβ are controlled by processes regulating the
nuclear shuttling of this kinase.

Acknowledgements

The authors wish to thank J. C. Stam (Molecular Cell Biology, Universiteit
Utrecht, The Netherlands) and B. Payrastre (INSERM unit 326, Toulouse, France)
for discussions and critical reading of the manuscript, and S. C. A. van den
Berg for his assistance during the production of monoclonal antibodies against
PI4K230.

Divecha, N., Banfic, H. and Irvine, R. F.
(
1991). The polyphosphoinositide cycle exists in the nuclei of
Swiss 3T3 cells under the control of a receptor (for IGF-1) in the plasma
membrane, and stimulation of the cycle increases nuclear diacylglycerol and
apparantly induces translocation of protein kinase C to the nucleus.
EMBO J.10,
3207
-3214.

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